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*
Gastrointestinal Unit, Department of Medicine, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114;
Division of Gastroenterology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02116; and
Division of Gastroenterology, Albany Medical College, Albany, NY 12208
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fractalkine (Neurotactin/NKAF) is a novel chemokine that is characterized by a CX3C spacing of the cysteine motif and has a unique membrane-bound structure (1, 2, 3). The domain organization includes a 37-residue intracellular tail, a short membrane-spanning region, and an extended, mucin-like stalk, which presents the N-terminal chemokine domain at the cell surface. This unique architecture represents a novel mechanism of cell adhesion that differs from soluble CXC, CC, and C chemokines, which have heparin-binding domains that may promote immobilization by cell surface proteoglycans or extracellular matrix components (4). Also, the juxtamembraneous part of fractalkine contains a dibasic motif (Thr-Arg-Arg-Gln) that can be cleaved, to yield chemoattractant soluble fractalkine. Thus, fractalkine represents a new class of chemokines that exhibits properties of both traditional chemokines and adhesion molecules (3). Fractalkine binds specifically to the chemokine receptor V28, now termed CX3CR1 (5, 6). CX3CR1 appears to be a highly specific receptor for fractalkine, since no additional chemokines that bind or compete for the receptor have been identified, with the exception of the HHV-8-derived virus chemokine vMIP-II (6, 7, 8). Surface expression of the CX3CR1 has been demonstrated in NK cells, monocytes, CD8+ T cells, and to a lesser extent in CD4+ T cells (5).
Intestinal intraepithelial lymphocytes
(iIEL)4 are thought to
represent a first-line defense against pathogens and may be critical
for intestinal integrity. Most iIEL express the
CD8+ phenotype with either CD8 heterodimeric
/ß-chains or homodimeric /-chains. iIEL exhibit a number of
important immunological functions, including cytotoxic activity
(9, 10); secretion of cytokines, including IL-2, IL-3,
IL-5, TNF-, TGF-ß, and IFN-
(11); and may regulate
epithelial cell proliferation and regeneration (12). In
addition, intestinal epithelial cells are capable of expressing MHC
class II molecules and can act as effective APCs that can induce and
activate T cells in vitro (13, 14, 15). Furthermore,
intestinal epithelial cells may selectively activate T cells with
suppressor function via CD1d molecule, which subsequently down-regulate
immune responses (16, 17, 18, 19, 20). Intestinal epithelial cells
also express IL-15 (21), which is a strong inducer of iIEL
activation and proliferation (22). Together, these
observations suggest that intestinal epithelial cells can provide
regulatory signals for T cells to maintain appropriate homeostasis in
the gastrointestinal tract. However, it is not clear how iIEL are
continually attracted toward intestinal epithelial cells and then
retained within the highly dynamic intestinal epithelium.
To determine the functional role of fractalkine expression in the intestinal mucosa, we have examined the source and function of the fractalkine-CX3CR1 ligand-receptor system in the healthy and diseased small intestinal mucosa.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA): anti-fractalkine C-18, directed against the extracellular domain of fractalkine; anti-fractalkine C-20, directed against the intracellular domain of fractalkine; anti-caveolin-1; and anti-E-cadherin. Anti-goat HRP was obtained from Amersham (Piscataway, NJ); anti-goat biotin, anti-mouse/rabbit biotin, and streptavidin-FITC were obtained from Dako (Carpenteria, CA). Streptavidin-PE, streptavidin-rhodamine, anti-rabbit biotin, anti-CD4 FITC, anti-CD8 biotin, and anti-CD8 PE used for FACS analysis were obtained from PharMingen (San Diego, CA). CX3CR1 expression constructs and antisera were a gift from P. M. Murphy (National Institute of Allergy and Infectious Disease, Bethesda, MD). Fractalkine, IL-1ß, and IL-2 were obtained from R&D Systems (Minneapolis, MN).
Cell culture
The human intestinal epithelial cell line T-84 was obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM/Ham F12 (50/50 v/v) medium (Cellgro; Mediatech, Herndon, VA) supplemented with 10% heat-activated FCS (Sigma), 1% penicillin, and streptomycin (Life Technologies, Gaithersburg, MD). Cells were grown at 37°C in a 5% CO2 atmosphere within a humidified incubator. Cells were always used for experiments at 70% confluence.
RT-PCR for the detection of CX3CR1 mRNA
The nucleotide sequence of CX3CR1 (GenBank accession number U28934) was analyzed and two primer generated: 5'-TTG AGT ACG ATG ATT TGG CTG A-3' and 5'-GGC TTT GGC TTT CTT GTG G-3'. PCR conditions were 30 s at 94°C, 30 s at 58°C, and 1 min at 72°C for 40 cycles.
Northern blot analysis
Total RNA was extracted from T-84 cells and intestinal biopsy specimens or resected intestinal tissues using Trizol reagent (Life Technologies), according to the manufacturer’s instructions. Crohn’s disease, ulcerative colitis, or normal intestinal control tissues were obtained from the tissue bank of the Center for the Study of Inflammatory Bowel Disease at Massachusetts General Hospital, according to the guidelines for the use of discarded human material at Massachusetts General Hospital. Resected tissue from three patients with Crohn’s disease and three specimens from control patients were analyzed.
Fractalkine cDNA was generated by RT-PCR using the following primers:
5'-ACT CTT GCC CAC CCT CAG C-3', and 5'-TGG AGA CGG GAG GCA CTC-3'. The
PCR products were subcloned into pCR 2.1 vector (Invitrogen, Carlsbad,
CA) and sequenced. cDNA probes were labeled with
[-32P]dCTP by a random hexamer priming
method using the Rediprime random primer labeling kit (Amersham Life
Science, Arlington Heights, IL). Membranes were hybridized in Quickhyb
solution (Stratagene, La Jolla, CA) at 68°C for 1 h. The
membranes were washed and the blots analyzed by autoradiography.
Expression of fractalkine mRNA was corrected for GAPDH expression and
quantitated densitometrically with the NIH Image 1.6.1 analysis
software.
Western blot analysis
Cells were placed in lysis buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 5 mM NaF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM PMSF, and 1 mM sodium-orthovanadate (protease/phosphatase-inhibitor mixture)). After 20 min on ice, cell lysates were cleared by centrifugation at 20,000 x g for 15 min at 4°C. Protein concentration in each sample was quantified by the Bradford method, and 20 µg protein was used for Western blot analysis. Proteins were separated by SDS-PAGE, using 10% Tris-glycine gels, and subsequently transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were incubated in blocking solution (1x TBS, 0.025% Tween 20 (TBS-T), 1% BSA) at room temperature for 1 h. After an overnight incubation with the first Ab (anti-fractalkine C-18 1:1000 or C-20 1:200 in blocking solution) at 4°C, membranes were washed four times in TBST. The HRP-labeled second Ab was dissolved (1:6000) in TBS-T, supplemented with 5% dry milk. After incubation with the second Ab for 1 h at room temperature, the membranes were washed four times in TBS-T and proteins were detected by the ECL method (Amersham, Piscataway, NJ), according to the instructions of the manufacturer. Western blots were quantitated densitometrically with NIH Image 1.6.1 analysis software.
Immunoprecipitation from T-84 cytosol protein and membrane protein fraction
T-84 cells were washed three times with ice-cold PBS, and 6 ml hypotonic lysis buffer (10 mM Tris, pH 7.4, 1 mM MgCl2, supplemented with the protease/phosphatase inhibitor mixture) was added to two 10-cm dishes. After incubation on ice for 20 min, the cells were disrupted by douncing for 15 times. By adding 5 M NaCl, iso-osmolar conditions were obtained. The cell lysate was cleared by centrifugation twice for 10 min at 1000 x g. The supernatant was then centrifuged at 100,000 x g for 35 min at 4°C. The supernatant (cytosolic fraction), was adjusted to a final concentration of 0.1% sodium deoxycholate, 0.1% SDS, and 0.1% Triton X-100. The pellet was solubilized in 1% sodium deoxycholate, 1% SDS, and 1% Triton X-100, and after an incubation for 20 min on ice, diluted to obtain identical conditions as in the cytosolic fraction. The lysate was dounced 10 times and cleared by a centrifugation at 10,000 x g for 15 min at 4°C, and referred to the membrane fraction. Equal (600 µg) amounts of protein, as determined by the Bradford method, were subjected to immunoprecipitation. After preclearing for 2 h with protein A/G beads (Calbiochem, La Jolla, CA), the lysates were incubated overnight with 2 µg/ml C 20 fractalkine Ab. After washing twice with washing buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.5% Triton X-100), the immunoprecipitate was denaturated by boiling in Laemmli buffer and subjected to Western blot analysis.
Isolation of detergent-insoluble glycolipid-enriched membrane microdomains
Glycolipid-enriched membrane microdomains or detergent-insoluble glycolipid rafts were isolated as described before (23) (24), with minor modifications. Four dishes of T-84 cells (10 cm diameter) were washed three times with ice-cold PBS, and the cells were solubilized in 1 ml of lysis buffer (25 mM MES, pH 6.8, 150 mM NaCl, 1% Triton X-100 supplemented with the protease/phosphatase inhibitor mixture). After incubation for 30 min on ice, the lysate was dounced gently 10 times and cleared by a centrifugation for 5 min at 1000 x g. The supernatant was transferred to the bottom of a centrifugation tube and adjusted to 40% sucrose with an equal volume of 80% sucrose in MBS (25 mM MES, pH 6.8, 150 mM NaCl supplemented with the protease/phosphatase inhibitor mixture). A sucrose step gradient with 30%, 25%, 20%, 15%, and 5% in MBS (2 ml each) was layered on top. This combination of sucrose concentrations gave the best resolution between 15% and 25% (data not shown). After a centrifugation in a SW 41 rotor (Beckman Coulter, Fullerton, CA) at 39.000 rpm for 14–16 h at 4°C, fractions of 1 ml each were taken, starting from the top (fraction 1), going to the bottom (fraction 12). For the preparation of cytosol and membranes, two dishes of T-84 cells (10 cm diameter) were three times washed with ice-cold PBS and taken up in 2 ml buffer A (20 mM Tris, pH 7.8, 1 mM EDTA, 0.25 M sucrose supplemented with the protease/phosphatase inhibitor mixture). The lysate was sheared 10 times and cleared by a centrifugation for 5 min at 1000 x g. One-fourth of the supernatant was taken for preparation of the cytosol. Four milliliters of buffer A were added, and a centrifugation for 60 min at 100,000 x g was performed. The middle, clear fraction referred to the cytosolic fraction. For membrane isolation, the remaining three-fourths of the cleared supernatant were layered on top of a 30% Percoll solution in buffer A, supplemented with 8.5% sucrose. After centrifugation for 30 min at 100,000 x g, the resulting interface, the membrane fraction, was isolated. Protein measurement of the cellular fractions was performed with BCA Protein Assay Reagent, according to the manufacturer’s instructions (Pierce, Rockford, IL). Equal amounts of protein were subjected to Western blot analysis, as described above.
Immunofluorescence staining for the detection of fractalkine
Intestinal biopsy specimen was obtained during diagnostic endoscopy after prior patients’ consent. The tissue was fixed with OCT-mounting medium (Miles, Elkhart, IN), immediately frozen in liquid nitrogen, and stored at -80°C until use. The tissue was cut at -22°C into 5-µm slices and put on positively charged glass slides and stored at -80°C until use. The slides were thawed for 5–10 min, dipped into acetone (-20°C) for 30 s, and air dried. T-84 cells were seeded at a density of 10,000 cells/ml and grown on coverslips (Lab-Tek, Naperville, IL) for 3 days. Cells were fixed as the intestinal biopsy specimens. After air drying, the cells or biopsy specimens, respectively, were blocked for 20 min in 5% (v/v) donkey serum (Santa Cruz Biotechnology) in PBS (blocking solution). All steps were conducted at room temperature in a humidified, light-protected chamber. After an incubation with anti-fractalkine C-20 (2.5 µg/ml), anti-cadherin, or anti-caveolin-1 (all from Santa Cruz Biotechnology, Santa Cruz, CA) in blocking solution for 2 h, slides were washed with PBS (2 x 2 min) and incubated with anti-goat biotin Ab in blocking solution (1:50) for 30 min. After an additional washing step, the slides were incubated with streptavidin-FITC (1:50) for 1 h. After washing, slides were mounted with Vectashield (Vector Laboratories, Burlingame, CA). Staining was analyzed with an IX70 Olympus-fluorescent microscope.
Isolation of iIEL and FACS analysis
Intestinal jejunal tissue was obtained during gastric bypass surgery for obesity. iIEL were isolated as described earlier (25). iIEL were either used freshly for FACS analysis or kept in culture, as described before (26). FACS analysis was performed, as described before (25), using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The anti-CX3CR1 Ab containing antisera was used at a dilution of 1/50; negative control experiments were performed with the preimmune serum also at a 1/50 dilution.
iIEL migration assay
iIEL were stimulated with IL-2 (10 ng/ml) for 3 days. Using a chemotaxis chamber (Neuroprobe, Cabin John, MD), the lower well was filled with medium with or without fractalkine, and, in some instances, anti-fractalkine Ab at concentrations indicated. A 5-µm polyvinylpyrrolidone-free polycarbonate membrane separated the lower from the upper well, which was filled with 50 µl medium, containing 100,000 iIEL. Each assay was performed in triplicate. The cells were allowed to migrate for 8 h at 37°C. Cells in the lower well were counted, and the migration index (migrated cells divided by the number of cells that migrated without fractalkine) was calculated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fractalkine mRNA expression has been found in different tissues,
including small intestinal and colonic tissue (3). The
latter prompted us to investigate whether fractalkine mRNA is expressed
in intestinal epithelial cells. Northern blot analysis demonstrated
that fractalkine mRNA is expressed in the intestinal epithelial cell
line T-84 (Fig. 1
). Incubation with the
NF-
B-inducing cytokine IL-1ß was able to induce a transient 4-fold
increase in basal steady state expression of fractalkine transcripts
within 2 h (Fig. 1).
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To determine whether the induction of fractalkine mRNA expression
in T-84 cells resulted in increased production of fractalkine protein,
Western blot and immunoprecipitation analysis were conducted with Abs
specifically directed against the chemokine domain and the
intracellular region of fractalkine. As demonstrated in Fig. 2, fractalkine was detected in T-84 cell
lysates running with a molecular size of 
95 kDa, which is consistent
with the previously described size of glycosylated fractalkine
(3). The basal expression of fractalkine in T-84 cells was
increased 2-fold within 12 h after incubation with 10 ng/ml
IL-1ß (Fig. 2, A and B). Fractalkine has been
demonstrated to be released in soluble form from its transmembrane
region, and we therefore determined whether fractalkine was released
from T-84 cells upon stimulation by IL-1ß. As demonstrated in Fig. 2, C and D, the increase of fractalkine protein
expression in T-84 cell lysates was followed by an elevation of soluble
fractalkine in the T-84 cell media supernatants. The basal expression
of fractalkine in T-84 cell media supernatants increased 5-fold over
24 h after induction by IL-1ß (Fig. 2, C and
D).
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, E and F, the anti-fractalkine Abs
hybridized to a protein band in the immunoprecipitates from the cytosol
as well as the transmembrane protein fraction, which corresponded to
the size of full-length glycosylated recombinant fractalkine (Fig. 2E, lane 5). More than 82% of the fractalkine
expressed in nonstimulated T-84 cells localized in the T-84 cell
membrane fraction. Stimulation with IL-1ß for 12 h increased the
fractalkine content in the cytosol fraction 2.5-fold and by 1.3-fold in
the membrane fraction (Fig. 2, E and F). Fractalkine is localized in caveolin-1 containing detergent-insoluble glycolipid-enriched membrane microdomains in T-84 cells
To further characterize the membrane compartment containing
fractalkine, we separated postnuclear membrane protein fractions by
sucrose density-gradient centrifugation. This method separates
detergent-insoluble functional distinct membrane fractions
characterized by their specific lipid composition (24, 27, 28). As demonstrated in Fig. 3A, fractalkine was present in
low density membrane fractions from T-84 cells. Fractalkine was present
in membrane fractions corresponding to sucrose concentrations of 15%
to 24%, with the highest concentration in the fraction corresponding
to 22% (Fig. 3A, lane 5). These low density
membrane fractions have been associated with detergent-insoluble
glycolipid-enriched membrane fractions or rafts (29). In
epithelial cells, these membrane fractions are characterized by the
presence of a distinct scaffolding protein caveolin (30).
As demonstrated in Fig. 3B, Western blot analysis of these
cellular compartments revealed that the expression of caveolin-1 was
restricted to same sucrose gradients as the fractalkine expression T-84
cells. Caveolin-1 concentration was maximal at apparently 22% sucrose
(Fig. 3B), consistent with a previous report
(31). This seems to be cell specific for T-84 cells, as
maximal amounts of caveolin are typically detected at sucrose
concentrations of 15–20% in other cell lines (24).
Collectively, these data indicate that fractalkine is largely
sequestered in a microdomain of T-84 cell membranes that display the
biophysical characteristics of caveolae (29).
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The human intestinal epithelial cell line T-84 provides a
well-established model for the assembly of intercellular junctions and
the development of apical-basolateral polarity (32).
Immunofluorescence staining revealed specific staining for fractalkine
in T-84 cells (Fig. 4A).
Fractalkine was mainly expressed in undifferentiated proliferating T-84
cells that are localized at the edges of monolayers with developed
apical-basolateral polarity (Fig. 4). Polarized T-84 cells still
retained some fractalkine expression, which appeared to be localized to
cell-cell contact regions. In contrast, fractalkine expression in
unpolarized T-84 cells demonstrated a strong granular cytoplasmic and
cell surface expression pattern (Fig. 4, A and
B). Because fractalkine demonstrated a strong enrichment in
caveolin-1-containing membrane fractions, we determined the expression
of caveolin-1 in polarized and unpolarized T-84 cells. As shown in Fig. 4, C and D, caveolin-1 was strongly expressed in
unpolarized T-84 cells. The specific expression pattern of fractalkine
and cavelolin-1 in unpolarized T-84 was not due to restricted access to
the basolateral surface of the polarized epithelial cells because
anti-E-cadherin Abs were able to detect E-cadherin on the surface
of unpolarized as well as at the basolateral surface of polarized T-84
cells (Fig. 4, E and F).
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To determine the potential target cells of fractalkine expression
by intestinal epithelial cell in the intestinal mucosa, we assessed
expression of CX3CR1 transcripts in RNA isolated
from lymphocyte population obtained from normal human small intestine.
As shown in Fig. 5, iIEL from three
independent cell isolations and two of three lamina propria lymphocyte
isolations demonstrated CX3CR1 mRNA expression.
In addition, CX3CR1 mRNA expression was detected
in the iIEL cell line EEI10.
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A, the freshly isolated human
iIEL consisted of 78% CD8-positive (Fig. 6A) and 7.6%
CD4-positive lymphocytes (Fig. 6B). A total of 48.5% of the
fresh isolated iIEL expressed the fractalkine receptor
CX3CR1 (Fig. 6C).
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To determine whether the CX3CR1 receptor
expressed on iIEL is able to mediate chemoattraction, the migratory
response of isolated human iIEL was determined in the chemotaxis
chamber multiwell system, as described (33), and expressed
in fold increase of the basal cell migration (Migration Index) (Fig. 7A). Since fresh isolated iIEL
do not migrate efficiently toward chemokine gradients, iIEL were
stimulated with IL-2 (10 ng/ml) for 3 days, as previously described
(34). As demonstrated in Fig. 7A, soluble
fractalkine induced the migration of IL-2-activated iIEL in a
dose-dependent fashion. Highest induction was found at a fractalkine
concentration of 1 ng/ml, and activity decreased at higher
concentrations. Similar dose-response curves have been reported for
RANTES in the induction of migration of iIEL (33). The
fractalkine-induced migration of iIEL could be significantly inhibited
in a dose-dependent fashion by Abs against the chemokine domain (C-18),
but not the carboxy terminus of fractalkine (C-20) (Fig. 7B,
p < 0.001). Flow cytometry confirmed the presence of
CX3CR1 on iIEL (Fig. 7C) after
incubation with IL-2. Furthermore, iIEL retained their CD8-expressing
phenotype during IL-2 activation (Fig. 7D). Together, these
experiments demonstrate that iIEL express functional
CX3CR1 receptors, and can be chemoattracted by
soluble fractalkine when activated by IL-2.
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Immunohistochemistry was utilized to determine whether fractalkine
is expressed in human normal or diseased small intestinal mucosa.
Frozen sections of normal and inflamed small intestinal mucosa (Fig. 8) were stained for the presence of
fractalkine with a combination of Abs against the intracellular (C-18)
and extracellular (C-20) regions of fractalkine, followed by incubation
with biotinylated anti-rabbit Abs and FITC-coupled streptavidin. As
demonstrated in Fig. 8A, intestinal epithelial cells in
normal small intestinal tissues stained positive for fractalkine. In
addition, a punctuated positive staining for fractalkine was present in
endothelial cells outlining small mucosal blood vessels (Fig. 8A, indicated by white arrows). Fractalkine expression was
highly up-regulated in endothelial cells outlining all small blood
vessels observed within the inflamed intestinal mucosa during Crohn’s
disease even in areas of the mucosa that did not show infiltration of
inflammatory cells (Fig. 8C). Furthermore, during intestinal
inflammation in Crohn’s disease, intestinal epithelial cells
demonstrated strong basolaterally positive staining for fractalkine
(Fig. 8, E and F). Together, these experiments
demonstrate that fractalkine can be expressed by small intestinal
epithelial cells and endothelial cells in the normal and inflamed small
intestine.
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To determine whether fractalkine expression is involved in
intestinal inflammation, we assessed fractalkine mRNA expression in
normal small intestine, as well as in intestinal tissues resected from
patients with active Crohn’s disease. As demonstrated in Fig. 9, fractalkine mRNA was expressed in the
normal small intestine (Fig. 9, lanes 1–3). Fractalkine
mRNA was significantly up-regulated in Crohn’s disease (Fig. 9, lanes 4 and 5). The expression of fractalkine
mRNA in small intestine increased from a mean density/area of 32 + 4.5
in normal intestine up to 85 + 16.2 in Crohn’s disease tissues after
correction for GAPDH expression (p <
0.01).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The detection of fractalkine transcripts in mRNA isolated from small and large intestine (3) prompted us to determine whether intestinal epithelial cells are a source of fractalkine mRNA and protein expression. Our experiments demonstrate that the human intestinal epithelial cell line T-84 is able to express membrane-bound and soluble fractalkine under the control of the inflammatory mediator IL-1ß, suggesting that fractalkine may be involved in directing intestinal epithelial cell-lymphocyte interactions as well as the attraction of lymphocytes into the intestinal lamina propria. Induction of fractalkine expression in intestinal epithelial cells may mediate long lasting effects. Whereas basal expression of fractalkine mRNA was maximally induced within 4 h after a single induction by IL-1ß, membrane-bound fractalkine protein levels increased over 12 h, and the amount of soluble fractalkine increased steadily over 24 h. Furthermore, the expression of fractalkine in intestinal epithelial cells was regulated during polarization and differentiation of T-84 cells. Immunohistochemistry demonstrated that fractalkine was highly expressed in nonpolarized T-84 cells, which are still able to migrate and to proliferate, whereas fractalkine expression decreased in T-84 cells after polarization and differentiation and localized to the basolateral cell surface. Fractalkine expression in undifferentiated and migrating intestinal epithelial cells may indicate a role of fractalkine in intestinal wound healing.
Membrane-bound fractalkine colocalized with caveolin-1 in a specialized membrane compartment characterized by its specific lipid composition, which allowed separation on a sucrose gradient from other membrane protein fractions. This indicates that fractalkine may be specifically expressed in caveolae on the surface of intestinal epithelial cells. Caveolae are vesicular invaginations of the plasma membrane characterized by the expression of the caveolin family of scaffolding proteins (30). Caveolins can assemble signaling molecules into preassembled signaling complexes, and an increasing number of signaling pathways have been shown to be associated with caveolae, such as those mediated by G protein-coupled receptors, the ras-raf pathway, and pathways involving the activation of src family tyrosine kinases (30). Signal transduction events important in cell-cell adhesion, such as integrin signaling, have been shown to be dependent on the presence of caveolins (45). Consistent with the expression of fractalkine in caveloae-like structures, the same nonpolarized T-84 cell population stained positive for the expression of caveolin-1. Targeting of fractalkine to glycolipid-enriched microdomains may be mediated by the myristylation of cysteines in the membrane or juxtamembrane region of fractalkine (C353, C367, and C386), analogous to the membrane targeting of LAT (linker for activation of T cells) (46). Furthermore, the punctuated expression pattern of fractalkine was observed in primary intestinal epithelial cells as well as on the surface of endothelial cells in the intestinal mucosa. The significance of the expression of fractalkine in specialized membrane fractions is not clear, but may aid the close attraction and interaction of leukocytes with signaling molecules on the surface of intestinal epithelial cells.
Cell populations regulated by fractalkine within the intestinal mucosa may include iIEL as well as lamina propria lymphocytes. CX3CR1 mRNA expression could be detected in RNA prepared from isolated human small intestinal IEL as well as lamina propria mononuclear cells. Flow cytometry revealed that a subpopulation of fresh isolated human iIEL expressed CX3CR1 on its surface. Consistent with these results, fractalkine was able to specifically chemoattract activated isolated human iIEL. In these experiments, isolated human iIEL required stimulation with IL-2 to migrate within fractalkine gradients. Although the IL-2-stimulated iIEL retained their CD8+ phenotype, it is not clear whether iIEL require additional signals by other chemokines or adhesion molecules to migrate in vivo. Fractalkine has been shown to mediate leukocyte capture and firm adhesion of nonstimulated and activated peripheral CD8+ T cells, monocytes, and CD16/56 NK cells (43). Although CX3CR1 mRNA was also expressed by IL-2-activated CD4+ T cells, these cells did not firmly adhere to immobilized fractalkine (5). Cultured human iIEL have been demonstrated to retain their capability to migrate into polarized epithelial cell monolayers in vitro, where they assume a subjunctional position, identical to that observed in vivo (47). This migration was partially inhibited by pertussis toxin, suggesting a potential mechanism for iIEL migration by chemokine receptor-mediated signaling (47). Furthermore, several chemokines, including IL-8 and RANTES, have been shown to be able to direct the migration of cultured intraepithelial lymphocytes (34). In these experiments, as in ours, cultured iIEL had to be activated by IL-2R signaling to migrate in response to chemokines. This may be due to the abrogation of chemokine receptor expression by PHA, used in the protocols to propagate iIEL, because PHA has been shown to down-regulate CX3CR1 expression (5) as well as CCR1 and CCR2 expression (48).
The mobilization of CD8+ T cells into intestinal
mucosa has been proposed to be cooperatively regulated by integrins and
chemokines (34, 49, 50). The
Eß7 integrin is
strongly expressed by iIEL (51, 52, 53).
Eß7 is involved in
iIEL binding to epithelial cells via interaction with E-cadherin
(54). The iIEL deficiency associated with a lack of
E or ß7 expression
would suggest that Eß7
is required for entry and/or retention of T cells into the intestinal
epithelium (55, 56). Because fractalkine has been shown to
mediate strong integrin-independent adhesion in vitro (5, 44), fractalkine expressed by intestinal epithelial cells may
not only attract iIEL toward the epithelium, but may also contribute to
the retention of iIEL within the intestinal epithelial cell layer.
In addition to the attraction of iIEL into the small intestinal mucosa,
fractalkine may play an important role during intestinal inflammation.
Fractalkine mRNA expression was strongly up-regulated in Crohn’s
disease mucosa, suggesting an involvement of the
fractalkine-CX3CR1 ligand receptor system in
recruitment of leukocytes during intestinal inflammation. Our data
demonstrate that both iIEL and LPL can express
CX3CR1 mRNA. The leukocyte cell populations
attracted by intestinal epithelial as well as endothelial cell-derived
fractalkine in intestinal inflammation need to be
determined. Fractalkine expression has been demonstrated in
HUVEC after induction with IL-1 or TNF-, and therefore a role of
fractalkine has been proposed in the regulation of inflammation
(3). However, to date, no direct demonstration of the
involvement of fractalkine in inflammatory disease has been reported.
The best evidence that fractalkine is involved in the attraction of
leukocytes during disease was demonstrated in experiments in which
fractalkine-mediated migration of granulocytes isolated after
Ab-induced glomerulonephritis could be inhibited by vMIP-2
(7).
The mechanism of lymphocyte migration into healthy and diseased intestinal mucosa is not well understood. Together, our studies support a model in which fractalkine may be involved in the attraction and retention of iIEL into the intestinal epithelium. Fractalkine expression within the intestinal mucosa may have an important function in intestinal inflammation. Endothelial-expressed fractalkine may initiate capture of lymphocytes by firm adhesion to the activated endothelium, and epithelial cell-derived soluble fractalkine may direct lymphocytes to the site of inflammation and tissue repair. Membrane-bound fractalkine expressed by intestinal epithelial cells may then help to retain iIEL within the regenerating intestinal epithelium.
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Footnotes |
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2 A.M. and L.J.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Hans-Christian Reinecker, Gastrointestinal Unit, Jackson Building, R711, Massachusetts General Hospital, 32 Fruit Street, Boston, MA 02114. E-mail address:
4 Abbreviations used in this paper: iIEL, intestinal intraepithelial lymphocyte; LPL, lamina propria lymphocyte.
Received for publication July 23, 1999. Accepted for publication January 10, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Y. Hoshino, D. B. Tse, G. Rochford, S. Prabhakar, S. Hoshino, N. Chitkara, K. Kuwabara, E. Ching, B. Raju, J. A. Gold, et al. Mycobacterium tuberculosis-Induced CXCR4 and Chemokine Expression Leads to Preferential X4 HIV-1 Replication in Human Macrophages J. Immunol., May 15, 2004; 172(10): 6251 - 6258. [Abstract] [Full Text] [PDF]
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X. Zhao, A. Sato, C. S. Dela Cruz, M. Linehan, A. Luegering, T. Kucharzik, A.-K. Shirakawa, G. Marquez, J. M. Farber, I. Williams, et al. CCL9 Is Secreted by the Follicle-Associated Epithelium and Recruits Dome Region Peyer's Patch CD11b+ Dendritic Cells J. Immunol., September 15, 2003; 171(6): 2797 - 2803. [Abstract] [Full Text] [PDF]
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C. Hundhausen, D. Misztela, T. A. Berkhout, N. Broadway, P. Saftig, K. Reiss, D. Hartmann, F. Fahrenholz, R. Postina, V. Matthews, et al. The disintegrin-like metalloproteinase ADAM10 is involved in constitutive cleavage of CX3CL1 (fractalkine) and regulates CX3CL1-mediated cell-cell adhesion Blood, August 15, 2003; 102(4): 1186 - 1195. [Abstract] [Full Text] [PDF]
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K. Shimoya, Q. Zhang, K. Tenma, Y. Ota, K. Hashimoto, Y. Shizusawa, T. Kimura, M. Koyama, and Y. Murata Fractalkine (FRK) levels in amniotic fluid and its production during pregnancy Mol. Hum. Reprod., February 1, 2003; 9(2): 97 - 101. [Abstract] [Full Text] [PDF]
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Q. Zhang, K. Shimoya, Y. Ohta, R. Chin, K. Tenma, S. Isaka, H. Nakamura, M. Koyama, C. Azuma, and Y. Murata Detection of fractalkine in human seminal plasma and its role in infertile patients Hum. Reprod., June 1, 2002; 17(6): 1560 - 1564. [Abstract] [Full Text] [PDF]
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A. Ludwig, T. Berkhout, K. Moores, P. Groot, and G. Chapman Fractalkine Is Expressed by Smooth Muscle Cells in Response to IFN-{gamma} and TNF-{alpha} and Is Modulated by Metalloproteinase Activity J. Immunol., January 15, 2002; 168(2): 604 - 612. [Abstract] [Full Text] [PDF]
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 K. Fujimoto, T. Imaizumi, H. Yoshida, S. Takanashi, K. Okumura, and K. Satoh Interferon-gamma Stimulates Fractalkine Expression in Human Bronchial Epithelial Cells and Regulates Mononuclear Cell Adherence Am. J. Respir. Cell Mol. Biol., August 1, 2001; 25(2): 233 - 238. [Abstract] [Full Text] [PDF]
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 P. S. Beisser, L. Laurent, J.-L. Virelizier, and S. Michelson Human Cytomegalovirus Chemokine Receptor Gene US28 Is Transcribed in Latently Infected THP-1 Monocytes J. Virol., July 1, 2001; 75(13): 5949 - 5957. [Abstract] [Full Text] [PDF]
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 D. N. Cook, S.-C. Chen, L. M. Sullivan, D. J. Manfra, M. T. Wiekowski, D. M. Prosser, G. Vassileva, and S. A. Lira Generation and Analysis of Mice Lacking the Chemokine Fractalkine Mol. Cell. Biol., May 1, 2001; 21(9): 3159 - 3165. [Abstract] [Full Text]
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B. Cambien, M. Pomeranz, H. Schmid-Antomarchi, M.-A. Millet, V. Breittmayer, B. Rossi, and A. Schmid-Alliana Signal transduction pathways involved in soluble fractalkine-induced monocytic cell adhesion Blood, April 1, 2001; 97(7): 2031 - 2037. [Abstract] [Full Text] [PDF]
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A. D. Lucas, N. Chadwick, B. F. Warren, D. P. Jewell, S. Gordon, F. Powrie, and D. R. Greaves The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo Am. J. Pathol., March 1, 2001; 158(3): 855 - 866. [Abstract] [Full Text] [PDF]
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C. A. Haskell, M. D. Cleary, and I. F. Charo Unique Role of the Chemokine Domain of Fractalkine in Cell Capture. KINETICS OF RECEPTOR DISSOCIATION CORRELATE WITH CELL ADHESION J. Biol. Chem., October 27, 2000; 275(44): 34183 - 34189. [Abstract] [Full Text] [PDF]
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J. K. Harrison, A. M. Fong, P. A. W. Swain, S. Chen, Y.-R. A. Yu, M. N. Salafranca, W. B. Greenleaf, T. Imai, and D. D. Patel Mutational Analysis of the Fractalkine Chemokine Domain. BASIC AMINO ACID RESIDUES DIFFERENTIALLY CONTRIBUTE TO CX3CR1 BINDING, SIGNALING, AND CELL ADHESION J. Biol. Chem., June 8, 2001; 276(24): 21632 - 21641. [Abstract] [Full Text] [PDF]
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R. Nishiyama, T. Sakaguchi, T. Kinugasa, X. Gu, R. P. MacDermott, D. K. Podolsky, and H.-C. Reinecker Interleukin-2 Receptor beta Subunit-dependent and -independent Regulation of Intestinal Epithelial Tight Junctions J. Biol. Chem., September 14, 2001; 276(38): 35571 - 35580. [Abstract] [Full Text] [PDF]
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C.-L. Tsou, C. A. Haskell, and I. F. Charo Tumor Necrosis Factor-alpha -converting Enzyme Mediates the Inducible Cleavage of Fractalkine J. Biol. Chem., November 21, 2001; 276(48): 44622 - 44626. [Abstract] [Full Text] [PDF]
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